Human embryonic stem (hES) cells have the remarkable ability to self-renew indefinitely or differentiate into any human cell type. Their abilty to differentiate into any tissue type offers great potential in regenerative medicine, drug discovery, and developmental biology. However, directing hES cells to a specific cell lineage has been a major challenge due to the lack of chemically defined tools to elucidate environmental cues that control self-renewal and differentiation. Human ES cells are keenly responsive to their physical environment, behaving differently under varying culture conditions. Specifically, the culture medium and the substratum composition can influence their decision to self-renew or differentiate. We have developed synthetic surfaces displaying peptides that bind to glycosaminoglycans (GAGs) and promote ES cell adhesion and self-renewal. These data indicate that GAGs are involved in ES cell self-renewal. GAGs are linear, anionic polysaccharides that can be displayed in the form of proteoglycans (PGs) on the cell surface. They are involved in cell-cell and cell-extracellular matrix (ECM) interactions. Of the GAG types, heparin and heparan sulfate (HS) have been implicated in ES cell regulation but there is little direct evidence for their involvement. The principal goal of this proposal is to understand the roles of GAGs in ES cell self-renewal and differentiation and to exploit this information to design synthetic surfaces to control ES cell fate. This proposal discusses the determination of functional roles of GAGs in hES cell self-renewal and differentiation, and the isolation and characterization of GAGs of ES and differentiated cells.